%0 Generic
%A Lampropoulos, Athanasios
%C Heidelberg
%D 2023
%F heidok:32788
%R 10.11588/heidok.00032788
%T Biochemical and molecular characterisation of the transcription factor WUSCHEL in Arabidopsis thaliana
%U https://archiv.ub.uni-heidelberg.de/volltextserver/32788/
%X Plants are autotroph sessile organisms that are characterised by extensive postembry- onic development. This continuous generation of new tissues and organs, which follows the germination of the plant embryo, is driven by multipotent stem cells that reside in special- ized tissues called meristems. The Shoot Apical Meristem (SAM) is the tissue where almost all aerial plant organs are generated. In the eudicot reference plant Arabidopsis thaliana, the stem cell pool of the SAM is located in the Central Zone (CZ) and is maintained by the cells of the adjacent Organising Centre (OC). The latter express the homeodomain transcrip- tion factor WUSCHEL (WUS), which is the key regulator of stem cell function in the SAM. WUS moves from the OC into the CZ through the plasmodesmata where it upregulates or represses its target genes. CLAVATA3 (CLV3), a gene activated by WUS in the CZ, encodes an oligopeptide that is secreted in the apoplastic space and subsequently moves back to the OC where it represses WUS, thereby establishing a negative feedback loop that controls stem cell fate.  The biological functions of WUS cannot be fully explained yet. However, it is known that WUS exerts its role in the SAM via physical interaction with other proteins. WUS acts as negative regulator of gene expression for a multitude of genes and this function is enhanced by its interaction with the transcriptional repressor TOPLESS (TPL). In addition, the interac- tion of WUS with transcription factor HAIRY MERISTEM 1 (HAM1) is a prerequisite for the spatially correct activation of CLV3 by WUS in the SAM. Therefore, I decided to ascertain the in vivo WUS complex with the goal of identifying novel WUS interactors in an attempt to elucidate WUS function.  To this end, I performed a yeast two-hybrid (Y2H) screening against an extensive library of transcription factors from A. thaliana. The interaction of a subset of these transcription fac- tors with WUS was further validated with Acceptor Photobleaching Förster Resonance En- ergy Transfer (AP-FRET) in planta. Among the candidates that I identified were WOX9 and ESR1, whose genetic interaction with WUS I dissected by means of generating and studying CRISPR mutants for the respective genes. Additionally, I performed co-immunoprecipitation reactions (co-IPs) of WUS fusion proteins that were expressed under the native WUS pro- moter in A. thaliana meristematic tissue or were overexpressed in Nicotiana benthamiana. The WUS complexes that I pulled down were further analysed with Mass Spectrometry. I also studied the effect of WUS phospho-PTMs by expressing the respective phospho-mutants and phospho-mimics in A. thaliana.